It has been revealed that various diseases are caused by increased generation of active oxygen species in vivo. Thus, dynamic analyses of active oxygen species in vivo are important for clarifying the causes, the states and the like of diseases. In the analyses of active oxygen species, a bio-imaging method by a fluorescent probe plays a predominant role. Since the generation of superoxide is the root cause of the production of all of active oxygen species, the analyses thereof are very important. However, examples of the fluorescent probe for superoxide include only hydroethidium (HE) (Scheme 1 described below) (about HE, see, for example, (1) M. Nakano, Cell. Mol. Neurobiol. 1998, 18, 565-579; (2) C. L. Murrant, M. B. Reid, Microsc. Res. Tech. 2001, 55, 236-248; (3) M. M. Tarpey, I. Fridovich, Circ. Res. 2001, 89, 224-236; (4) T. Münzel, I. B. Afanas'ev, A. L. Klescchyov, D. G. Harrison, Arterioscler. Thromb. Vasc. Biol. 2002, 22, 1761-1768; (5) M. D. Esposti, Methods, 2002, 26, 335-340; (6) G. Rothe, G. Valet, J. Leukoc. Biol. 1990, 47, 440-448; (7) W. O. Carter, P. K. Narayanan, J. P. Robinson, J. Leukoc. Biol. 1994, 55, 253-258; (8) V. P. Bindokas, J. Jordán, C. C. Lee, R. J. Miller, J. Neurosci. 1996, 16, 1324-1336; (9) A. B. Al-Mehdi, H. Shuman, A. B. Fisher, Am. J. Pysiol. 1997, 272, L294-L300; (10) L. Benov, L. Sztejnberg, I. Fridovich, Free Radic. Biol. Med. 1998, 25, 826-831, etc.).

Moreover, a fluorescence mechanism of HE is based on an oxidation reaction caused by superoxide, and thus has a problem in specificity (selectivity). That is, because many active oxygen species work as oxidizers, HE is oxidized also by other active oxygen species, and the magnitude of the oxidation is peroxynitrite>hydroxyl radical>superoxide>hydrogen peroxide, in this order. Furthermore, it is known that HE also is oxidized by cytochrome c. Therefore, it is thought that a fluorescence response obtained by using HE should be used, not as an indicator of an amount of generated superoxide, but as an indicator of a “total amount of oxidizers generated in vivo including active oxygen species”. Whereas, examples of a probe based on a reducing power of superoxide include nitroblue tetrazolium (NBT). NTB is reduced by superoxide so as to be transformed into blue diformazane. However, an absorption probe of NBT has various problems that it is reduced by various kinds of reductase such as NOS, and the produced diformazane is transformed into NBT by disproportionation or oxidation, and moreover, the absorption probe of NBT cannot be used for leading-edge fluorescence analysis methods such as flow cytometry and confocal laser microscopes. With such a background, development of a fluorescent probe that responds to superoxide with high selectivity is required in the light of the cytophysiology and the like.
Whereas, assay methods including an acetyl cholinesterase (AChE) assay are important in the biochemical and medical fields of technologies. It is thought that a compound (a mercapto group-detecting compound) that can detect a compound having a mercapto group (a thiol group) can be used effectively for such essay methods. Several compounds having mercapto group-detectability actually have been developed so far, and they mainly are classified into the following three groups of types 1 to 3: the type 1 represents labeling reagents of fluorescent compounds; the type 2 represents fluorescence derivatizing agents for mercapto groups; and the type 3 represents coloring reagents based on reactions with mercapto groups. Examples of the compounds belonging to these types 1 to 3 and the general description of their mercapto group-detecting mechanisms will be shown respectively in Scheme 2 below.

The reagent belonging to the type 1 such as the compound 9 is used only for synthesizing fluorescence-labeled protein or nucleic acid, and has a below-stated problem if being used for an AChE assay and the like. Firstly, the compound 9 is a fluorescent compound similar to its product 10. Accordingly, in the case of conducting an AChE assay or measurement of an AChE inhibitory activity by using the compound 9, the operational processes thereof are complicated because of the necessity to separate 9 and 10 after an enzyme reaction. Moreover, since maleimide that is a reaction part in 9 with a mercapto group reacts also with other nucleophilic reagents such as amines and alcohols, the reagent of the type 1 has a problem in specificity (selectivity).
Next, the reagent belonging to the type 2 such as the compound 11 is used only for a labeling agent for a separation analysis. The reagent 11 of the type 2 is non-fluorescent, and provides 12 that is a fluorochrome by reacting with a mercapto group. Thus, the operational process for separation with the reagent after the reaction is not necessary, unlike the case of 9. However, 11 also reacts with various nucleophilic agents so as to provide fluorescent compounds that are similar to 12, and thus has a problem if being used for an AChE assay and the like in terms of the specificity. However, 11 reacting with various nucleophilic agents is advantageous to be used as a labeling agent for the nucleophilic agents, and thus is used favorably for a labeling agent for HPLC analyses of amines and thiols.
The reagent 13 (an Ellman's reagent) of type 3 actually is used for an AChE assay. However, 13 has a problem of its poor stability in an aqueous solution, that is, low sensitivity caused by a high blank response.
As another example of the compound except the reagents of the types 1 to 3 that selectively reacts with thiol, a sulfonamide compound 15 that causes a deprotecting reaction with thiol is reported (Fukuyama, T., et al., Tetrahedron Lett., 1997, 38, 5831-5834). It also is reported that a nucleophilic aromatic substitution reaction of the compound 15 proceeds more smoothly with a thiol compound than with an amino compound. More specifically, in the case of an amino compound, it is necessary to use an amount thereof much larger than that of 15 and requires a long period of reaction time. However, none of the compound 15 and its product shows fluorescence, and thus they cannot be expected to be used as a thiol-detecting compound at all.

As mentioned above, in the light of the application of an AChE assay method using acetyl thiocholine as a substrate, as a more common method, the development of a new mercapto group-detecting compound to replace the compound 13 and the like has been required.